PROJECT SUMMARY: Thyrold hormone receptors (TRs) are ligand-dependent, transcriptional regulators of metabolism. TRs repress gene expression in the absence of hormone, which is paradigmatic for other nuclear receptors (NRs) that function as repressors in the unliganded state. Repression is mediated by interaction with corepressors NCoR (Nuclear Receptor Corepressor) and SMRT (Silencing Mediator of Retinoid and Thyroid receptors), which exist in stoichiometric association with the chromatin-modifying enzyme, histone deacetylase 3 (HDACS). HDAC3 derives its catalytic activity from interacting with the unique Deacetylase Activation Domain (DAD) of NCoR/SMRT. The DAD-dependent NCoR/SMRT-HDACS complex is critical for repression by TR and other NRs in vitro, but the role of this interaction in vivo is unknown. We propose to use gene targeting and mouse phenotyping to understand the physiological relevance of the HDACS corepressor complexes. We hypothesize that these DAD-dependent interactions are very important, and affect distinct physiological pathways involving NRs. Specific Aim 1 is to determine the physiological functions of the NCoR and SMRT DAD domains. We have demonstrated that the DADs are required for the enzyme activity of HDACS in vivo, using novel mouse models in loss of function mutations have been knocked into the DAD domains of NCoR and SMRT. We hypothesize that this epigenomic modifying activity underlies some but not all of the biological activities of HDACS. This will be tested by thorough metabolic and molecular phenotyping of the N-DADm, S-DADm, and NS-DADm mice. Specific Aim 2 is to determine the physiological, tissue-specific functions of HDACS. The adipose and skeletal muscle-specific knockouts will be rigorously phenotyped both physiologically and molecularly. ChlP-seq and transcriptomic analysis will be performed in each tissue at ZT10 and ZT22, given the proven circadian role of HDACS in other tissues such as liver. Specific Aim 3 is to understand the determinants of tissue-specific NR corepressor functions corepressors on a genome-wide level. NR corepressor complexes are expressed in every cell type, yet have highly cell-specific functions that are manifest by tissue-specific cistromes. We hypothesize that this is due to recruitment of NCoR, SMRT, and HDACS by NRs and tissue-specific transcription factors (TFs). This will be tested by rigorous bioinformatic analysis of the cistromes of NCoR, SMRT, and HDACS in liver, white and brown adipose tissue, BAT, liver, heart, skeletal muscle, and beta cells, at different times of day, under different dietary and hormonal conditions. Together, these innovative and unique studies will elucidate mechanisms regulating transcription repression by TR and other NRs in a physiological context. The insights gained from this work will shed new light on the transcriptional and epigenetic control of key biological pathways, including metabolism and inflammation, with the potential to lead to new and deeper insights into metabolic disorders, such as obfesity, diabetes, and cardiovascular disease.